Cystic fibrosis (CF) is the most common lethal autosomal recessive genetic disorder that affects the Caucasian population. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR), and is characterized by a viscous airway surface liquid (ASL) that impairs mucociliary function and facilitates bacterial infection. The molecular mechanisms by which these symptoms result from CFTR malfunction are unclear. We hypothesized that expression and secretion of innate immune proteins is altered in CF ASL.
We sought to use cell culture models in which the only source of secreted proteins was differentiated airway epithelium. Since CFTR localizes to the apical surface of airway submucosal glands (SMG) and ciliated epithelium, cell culture models that recapitulate two parts of respiratory tract epithelium were studied: 1) SMG acini and 2) mucociliary epithelium.
We developed a three-dimensional system wherein CF (ΔF508/ΔF508) and non-CF human bronchial epithelial (HBE) cells differentiated on Matrigel into polarized glandular acini with mature lumens by two weeks with no significant variability in size. Bronchial acini expressed and secreted SMG proteins, MUC5B and lysozyme, at day 22, and exhibited vectorial secretions that were collected along with acinar cell lysates. Proteome profiling demonstrated unique protein signatures for each cellular space. However, abundant contaminating proteins from Matrigel and growth media were identified. Therefore, the ALI cell culture model of airway epithelium was chosen for quantitative proteomic comparison of CF and non-CF HBE apical secretions because the protein-rich media does not contact the apical surface.
CF and non-CF HBE cells were labeled by stable isotope labeling with amino acids in cell culture and differentiated at ALI. LC-MS/MS and bioinformatic analysis identified seventy-one proteins with altered levels in CF secretions (+/−1.5 fold-change; p-value<0.05). Validation with antibody based biochemical assays demonstrated increased levels of MUC5AC, MUC5B, fibronectin and MMP9, and increased proteolysis/activation of complement C3, in CF secretions. Overall, the function of altered proteins in the CF secretome is indicative of an airway epithelium in a state of repair and altered immunity in the absence of infection, suggesting the downstream consequences of mutated CFTR in CF airways set the stage for chronic inflammation and infection.
|Advisor:||Rose, Mary C.|
|Commitee:||Brown, Kristy J., Colberg-Poley, Anamaris M., Freishtat, Robert J., Hathout, Yetrib, Nagaraju, Kanneboyina|
|School:||The George Washington University|
|Department:||Biochemistry and Molecular Biology|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 75/01(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Systematic, Bioinformatics|
|Keywords:||Cystic fibrosis, Human bronchial epithelial cells, Quantitative proteomics, Secreted proteins, Three-dimensional culture|
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